Subclinical dose irradiation triggers human breast cancer migration via mitochondrial reactive oxygen species

Background Despite technological advances in radiotherapy, cancer cells at the tumor margin and in diffusive infiltrates can receive subcytotoxic doses of photons. Even if only a minority of cancer cells are concerned, phenotypic consequences could be important considering that mitochondrial DNA (mtDNA) is a primary target of radiation and that damage to mtDNA can persist. In turn, mitochondrial dysfunction associated with enhanced mitochondrial ROS (mtROS) production could promote cancer cell migration out of the irradiation field in a natural attempt to escape therapy. In this study, using MCF7 and MDA-MB-231 human breast cancer cells as models, we aimed to elucidate the molecular mechanisms supporting a mitochondrial contribution to cancer cell migration induced by subclinical doses of irradiation (< 2 Gy). Methods Mitochondrial dysfunction was tested using mtDNA multiplex PCR, oximetry, and ROS-sensitive fluorescent reporters. Migration was tested in transwells 48 h after irradiation in the presence or absence of inhibitors targeting specific ROS or downstream effectors. Among tested inhibitors, we designed a mitochondria-targeted version of human catalase (mtCAT) to selectively inactivate mitochondrial H2O2. Results Photon irradiation at subclinical doses (0.5 Gy for MCF7 and 0.125 Gy for MDA-MB-231 cells) sequentially affected mtDNA levels and/or integrity, increased mtROS production, increased MAP2K1/MEK1 gene expression, activated ROS-sensitive transcription factors NF-κB and AP1 and stimulated breast cancer cell migration. Targeting mtROS pharmacologically by MitoQ or genetically by mtCAT expression mitigated migration induced by a subclinical dose of irradiation. Conclusion Subclinical doses of photon irradiation promote human breast cancer migration, which can be countered by selectively targeting mtROS. Supplementary Information The online version contains supplementary material available at 10.1186/s40170-024-00347-1.


Introduction
X-ray radiotherapy with or without (neo)adjuvant hormonal therapy and/or chemotherapy is a gold standard treatment option for women with breast cancer.However, treatment efficacy is limited by intrinsic and acquired radioresistance, an escape mechanism embroiled in intensive research [1][2][3][4][5][6].A less studied escape mechanism is the potential for radiotherapy to stimulate cancer cell migration based on a natural tentative of cancer cells to leave the irradiation field.Modern advances in intensity-modulated radiotherapy [7] limit this possibility, as precise and accurate photon dose deposition can be achieved for most cancer cells within primary breast tumors.However, dose deposition is less accurate at the tumor margin and for cancer cells in diffusive infiltrates [8].This is inherent to limited imaging resolution especially under breathing movements, making it difficult to precisely localize and target the tumor margin [9][10][11].Peripheral cancer cells may thus receive subcytotoxic doses of photons, adapt, and escape.
To acquire migratory capacities is a first and key step towards exiting the irradiation field.While doses used for fractionated radiotherapy (1.8 to 2 Gy for conventional fractionation, 1.5 Gy bid for hypofractionation in inflammatory breast cancer, and ≥ 2.1 Gy for postoperative hypofractionation [12,13]) are generally (but not always) detrimental to migration for radiosensitive cancer cells [14,15], lower subcytotoxic doses can induce breast cancer cell proliferation [16], migration and invasion [17,18].With respect to breast cancer cell migration, the main mechanism identified to date is induction of an epithelial-to-mesenchymal transition [17,18], with N-cadherin, vimentin, focal adhesion kinase signaling and nuclear β-catenin contributing to the migratory phenotype [19].
In the present study, we tested the hypothesis that mitochondria within breast cancer cells are a promigratory signaling hub activated by subclinical doses of ionizing radiation (defined here as < 2 Gy) based on two paradigms.The first is the increased vulnerability of mitochondrial DNA (mtDNA) to irradiation, due to the fact that mtDNA is principally composed of coding regions, is not protected by histones, and has limited repair capabilities compared to nuclear DNA [20].Irradiation-induced mitochondrial dysfunction could thus persist and propagate until full mitochondrial turnover (fission, mitophagy, mitochondrial biogenesis and fusion) [21].The second paradigm is that an increased subcytotoxic production of mitochondrial reactive oxygen species (mtROS) is sufficient to trigger breast cancer cell migration [22,23].mtROS mainly originate from the mitochondrial electron transport chain (ETC), and either an increased or a decreased ETC activity following bottlenecking damage results in enhanced electron leak [22].Leaking electrons create mtROS, which collectively promote cancer cell migration by activating redox-sensitive effectors, including the transforming growth factor β (TGFβ) pathway [22].Mitochondrial dysfunction can thus support sustained cancer cell migration, but whether subclinical doses of radiotherapy facilitate this event in breast cancer cells is currently unknown.We explored and validated this possibility in vitro using two different types of human breast cancer cells (luminal A and triplenegative).We report that mtROS can be genetically and pharmacologically targeted to block the gain in migration induced by subclinical doses of radiation.

Irradiation
Adherent cells in culture dishes were irradiated at a dose rate of 0.8 Gy/min using an IBL-637 137 Cs photon irradiator (Gamma Service Medica).They were allowed to recover for 24 h before any other experimental intervention.

Migration and invasion
Migration and invasion were assayed in 24-well transwell plates with 8.0 μm pore size inserts (Corning, catalog #353,097) with 0.2% (MDA-MB-231) or 10% (MCF7) FBS as chemoattractant, as previously reported [24].After 24 h of migration or invasion in the presence of tested pharmacological agents, cells at the bottom of the insert were fixed with 4% paraformaldehyde (PFA) for 10 min, washed twice with PBS, and stained with 0.5% crystal violet for 2 h.Remaining cells at the top of the insert were removed with a cotton swab.Pictures were taken at 5x magnification on a Zeiss Axiovert S100 microscope and quantified using QuPath version 0.2.3 (University of Edinburgh).All results are expressed as % of the basal migration of untreated cells.

mtDNA quantification
The quantification of total and deleted mtDNA (common 4977 bp deletion) was performed using multiplex PCR on a ViiA 7 Real-Time PCR system (Applied Biosystems), using a previously described protocol [25].Briefly, primers encoding sequences from the minor arc (total mtDNA) and major arc (damaged mtDNA) of the mitochondrial genome were amplified and quantified with FAM and NED fluorescent probes, respectively.Data were normalized to nuclear DNA levels (nuclear gene β2M detected with the VIC probe).

Oximetry
Cellular oxygen consumption rates (OCRs) were determined using a Seahorse XFe96 bioenergetic analyzer (Agilent Technologies), according to manufacturer's protocol.Briefly, 24 h after irradiation or sham, 10,000 MCF7 or 5,000 MDA-MB-231 cells were seeded in their routine culture medium in XFe96 culture plates, treated pharmacologically as indicated, and left to adhere for 24 h.Cells where then assayed in CO 2 -free DMEM containing 10 mM glucose, 2 mM glutamine, 1.85 g/L NaCl, 3 mg/L phenol red, pH 7.4, using the XF cell MitoStress kit (Agilent Technologies) in the presence of indicated pharmacological modulators.Mitochondrial OCR (mtOCR) was calculated as the difference between basal OCR and nonmitochondrial OCR measured upon full ETC inhibition by 0.5 µM of Complex I inhibitor rotenone + 0.5 µM of Complex III inhibitor antimycin A.

Glucose and lactate measurements
Glucose uptake and lactate secretion rates were determined by measuring glucose and lactate concentrations in cell medium 48 h after treatment using a CMA600 enzymatic analyzer (Aurora Borealis), as previously described [26].

Generation of mitochondrial H 2 O 2
To selectively generate H 2 O 2 in mitochondria, cells were transfected with a mtHyPer-D-amino acid oxidase (DAAO) plasmid (Addgene, catalog #168,304) [29] using lipofectamine 3000.Within mitochondria, flavoenzyme DAAO generates H 2 O 2 by catalyzing the conversion of exogenously supplied D-alanine, but not L-alanine, to pyruvate, and the fluorescent sensor HyPer selectively reports on mtH 2 O 2 levels [30], which were measured on a Spectramax i3x spectrophotometer equipped with a MiniMax imaging cytometer.

Apoptosis detection
Apoptosis was detected using previously disclosed protocols that are detailed in the Supplementary Methods.

Statistical analyses
All results are expressed as means ± standard error of the mean (SEM) for n independent observations.Error bars are sometimes smaller than symbols.Outliers were identified using Dixon's Q test.Data were analyzed using GraphPad Prism 8.4.3.Student's t test and one-way ANOVA were used where appropriate.P < 0.05 was considered to be statistically significant.

Irradiation at subclinical doses promotes human breast cancer cell migration and mtROS production
To test whether subclinical doses of irradiation could promote cancer cell migration, luminal A MCF7 and triple-negative MDA-MB-231 human breast cancer cells were irradiated at photon doses ranging from 0.125 to 2 Gy (2 Gy being a reference clinical dose [12]) and assayed in transwells with FBS as chemoattractant.Peaks in migration were detected 48 h after 0.5 Gy for MCF7 (+ 42.6 ± 12.3%) and 48 h after 0.125 Gy for MDA-MB-231 (+ 24.5 ± 13.7%) cells (Fig. 1a).These doses were subcytotoxic (Fig. 1b).They did not induce breast cancer cell invasion in transwells (Figure S1).Metabolically, photon irradiation dose-dependently increased the mtOCR of MCF7 cells, which peaked 48 h after 0.5 Gy (Fig. 1c).Conversely, MDA-MB-231 cell mtOCR was significantly decreased 48 h after 0.125 Gy.The glycolytic rate (lactate/glucose ratio) of the two cell lines was unchanged (Fig. 1d).The increased mtOCR of MCF7 cells could be explained by an increased mitochondrial quality (more undamaged mtDNA), whereas the decreased mtOCR of MDA-MB-231 cells was associated with persistent mtDNA damage (common deletion) despite an increased total mtDNA content (Fig. 1e).
With respect to irradiation doses, maximal effects on mtOCR (Fig. 1c) correlated with maximal effects on migration (Fig. 1a).In the same conditions, mtROS levels were induced in MCF7 cells for doses ranging from 0.25 to 1 Gy (Fig. 1f ).Actually, mtROS, mtH 2 O 2 and total ROS levels were all significantly increased 48 h after 0.5 Gy (Fig. 1g).In MDA-MB-231 cells, the migration and mtOCR peaks observed at 0.125 Gy closely corresponded to the maximal mtROS levels also observed at 0.125 Gy (Fig. 1h).mtROS, mtH 2 O 2 and total ROS levels were all significantly increased 48 h after 0.125 Gy (Fig. 1i).This indicated that mtROS could participate in the migratory response of breast cancer cells irradiated at subclinical doses.

Targeting mtROS inhibits irradiation-induced breast cancer cell migration
Whether migration induced by subclinical doses of irradiation depends on mtROS production was tested using N-acetyl-L-cysteine (NAC, a general antioxidant) and MitoQ (selectively targeting mtROS) [31].The two antioxidants inhibited basal and irradiation-induced breast cancer cell migration, with NAC being more effective for MCF7 and MitoQ for MDA-MB-231 cells (Fig. 2a).In general, mitochondrial superoxide has a very short halflife, as it is rapidly converted to H 2 O 2 by mitochondrial superoxide dismutase 2 (SOD2) [32].Whether mtH 2 O 2 is involved in the breast cancer cell migration induced by subclinical doses of irradiation was tested using a mitochondria-targeted version of human catalase (mtCAT) (Figure S2 and Supplementary Methods), which effectively blocked irradiation-induced mtH 2 O 2 production by both cell lines (Fig. 2b).Downstream, mtCAT completely inhibited irradiation-induced MCF7 and MDA-MB-231 cancer cell migration (Fig. 2c).Collectively, we concluded at this stage that subclinical doses of radiation trigger human breast cancer cell migration by inducing long-lasting ETC dysfunction, resulting in enhanced mitochondrial superoxide and mtH 2 O 2 production.

Generating H 2 O 2 within mitochondria stimulates breast cancer cell migration
A corollary hypothesis was that elevating mtH 2 O 2 levels could be sufficient to induce breast cancer cell migration, which was tested using a mtDAAO-HyPer mitochondria-targeted system [29] (Fig. 3a).In the presence of D-alanine, DAAO increased mtH 2 O 2 levels in MCF7 and MDA-MB-231 cells (Fig. 3b), which promoted their migration (Fig. 3c).Combining D-alanine supplementation and subclinical doses of radiation further increased mtH 2 O 2 levels (Fig. 3b), but not cancer cell migration (Fig. 3c).We then tested if additional mtH 2 O 2 generated by mtDAAO-HyPer exacerbated promigratory cell response by inducing cell death pathways; however both cell lines exhibited no change in cytochrome c release, caspase cleavage, or cellular apoptosis/necrosis, as measured by Annexin V/PI staining (Figure S3).

Transcription factors AP1 and NF-κB participate in breast cancer cell migration induced by subclinical doses of radiation
The mitogen-activated kinase (MAPK) pathway has been suggested to promote cancer cell migration in a ROSsensitive manner [33].Accordingly, MAP2K1/MEK1 expression was induced 48 h after a 0.5 Gy dose delivery to MCF7 cells (Fig. 4a).This response was inhibited by MitoQ, linking irradiation-induced mtROS production to MAPK signaling in these cells.However, subclinical dose delivery to MDA-231 cells comparatively repressed MAP2K1/MEK1 expression independently of the presence of MitoQ (Fig. 4a), indicating that mtROS signaling is multifactorial.We therefore decided to focus on ROSsensitive transcription factors.
Downstream of the MAPK pathway and of several other ROS-sensitive pathways [34], transcription factors AP1 and NF-κB are known to be ROS-inducible  B, C) [35][36][37] and to promote cancer cell migration [36,38], but whether they could be activated by mtROS 48 h after subclinical dose irradiation was unknown.Inhibiting AP1 with T-55241 reduced the basal migration and blunted the subclinical radiation-induced gain in migration of both MCF7 and MDA-MB-231 cells (Fig. 4c).Similarly, inhibiting the transcriptional activity of NF-κB with IKK inhibitor BMS-345541 blocked basal migration and radiation-induced migration of the two cell lines (Fig. 4b), indicating that both AP1 and NF-κB and participate in the irradiation-induced promigratory phenotype in breast cancer cells.Their relative contribution was further evaluated in our cell models using fluorescent reporters of their transcriptional activities.In MCF7 cells, a 0.5 Gy irradiation reduced AP1 activity (Fig. 4d) but increased NF-κB activity (Fig. 4e), and the two answers were blocked by MitoQ.Comparatively, a 0.125 Gy irradiation activated AP1 (Fig. 4d) but did not modify NF-κB activity (Fig. 4e) in MDA-MB-231 cells.AP1 activation did not occur in the presence of MitoQ (Fig. 4d).Irradiation at subclinical doses can thus activate mtROS-sensitive promigratory transcription factors, but their nature differed across different human breast cancer cell lines (Fig. 4f ).

Targeting mtROS does not reduce the cell killing therapeutic activity of ionizing radiation
We finally aimed to provide some relevance to our observations with respect to photon radiotherapy, generally delivered in 5 fractions per week in clinical settings.One and two fractions of 0.5 Gy induced MCF7 cell migration, but this gain was lost with additional fractions (Figure S4a).Comparatively, MDA-MB-231 cell migration was increasingly induced, reaching a maximum of ~ 4-fold from 2 to 5 fractions.
While subclinical doses of radiation were used throughout this study to model dose deposition at and beyond the tumor margin, most breast cancer cells in  [12].MitoQ did not interfere with irradiation-induced cell killing at 2 Gy (Figure S4b), supporting its potential use as an adjuvant treatment with photon radiotherapy to counter breast cancer cell migration induced by subclinical doses of irradiation.

Discussion
In this study, we tested whether long-lasting mitochondrial alterations could promote human breast cancer cell migration.To avoid focusing on idiosyncrasies, we intentionally used two very different human breast cancer cell lines representing luminal A and triple-negative subtypes.They further represent contrasting metabolic archetypes, as MCF7 cells are oxidative whereas MDA-MB-cells are glycolytic in vitro [39].We identified a sequence of events accounting for migration induced by subclinical doses of radiation (< 2 Gy), commencing with the induction of mitochondrial dysfunction, mtROS production and subsequent activation of redox-sensitive transcription factors.mtROS generation was a shared response between both cell lines.It was still detected 48 h after irradiation and was, thereby, lending itself to pharmacological or genetic repression after irradiationinduced migration.
Our results show that oxidative MCF7 cells demanded a higher irradiation dose to optimally trigger migration than glycolytic MDA-MB-231.This phenomenon can be explained by both increased mitochondrial fitness and lower basal mtROS [40].Nevertheless, the correlation that we observed between the irradiation dose needed to trigger optimal migration and changes in mtOCR and increased mtROS levels was striking, even if the nature of the mitochondrial dysfunction differed.In the case of MCF7, we postulate that increased undamaged mtDNA content 48 h after irradiation could be the result of an increased mitochondrial turnover and, therefore, mitochondrial abundance.This would logically lead to increased mtROS production via increased cell respiration, which is known to be intrinsically coupled with electron leak from the ETC [41].Of note, the resulting acquisition of a migratory phenotype depends on (mt)ROS, as shown by the inhibitory effects of NAC and MitoQ, but is also likely modulated by repressors and/or damage to the migratory machinery at irradiation doses > 0.5 Gy [42].This would explain why increased mtROS production was not always sufficient to trigger MCF7 cell migration.In contrast to MCF7, glycolytic MDA-MB-231 cells displayed an increase in persistent mtDNA damage 48 h after a 0.125 Gy irradiation, associated with a drop in mtOCR despite increased mtDNA content.Here, we suggest a compensatory response to increase mitochondrial biogenesis accompanied by a delay in the clearance of damaged mitochondria.Preserved mtOCR at irradiation doses higher that 0.125 Gy may be explained by the activation of cellular antioxidant defenses above a low dose threshold, as previously proposed by others [43].This is supported by our observation that an increase in mtH 2 O 2 by mtDAAO-HyPer did not further induce migration in either cell line, nor did it induce an increase in cytochrome c release or apoptosis/necrosis (Figure S3), which most likely implicates that continual sustained mtH 2 O 2 generation was not enough to overwhelm cellular antioxidant defense.In the case of MDA-MB-231 cells, increased mtROS production can be directly attributed to mitochondrial defects known to be associated with increased electron leak upon bottlenecking ETC damage [22].This could then lead to reverse ETC flux associated with Complex I electron leakage [44].The mitochondrial response of breast cancer cells to subclinical doses of radiation is summarized in Fig. 4g.Of note, although we posit mtDNA alterations as an initial trigger to increase mtROS levels, an additional contribution of mitochondrial content [45], swelling versus shrinkage [46] and fission versus fusion dynamics [47] is possible.
When electrons leak from the ETC, mitochondrial superoxide is formed followed by mitochondrial H 2 O 2 generation.With a longer half-life, H 2 O 2 can permeate the mitochondrial membrane [41] and act as a redox signal to activate ROS-sensitive promigratory pathways [48].c-Src kinase belongs to one of these pathways: its oxidation activates the TGFβ pathway [22,49] resulting in the upregulation of the focal adhesion kinase Pyk2 that remodels the cytoskeleton for migration.In breast cancer cells, we further report that subclinical doses of irradiation activate redox-sensitive transcription factors AP1 and NF-κB that cooperate to induce migration.The process would logically depend on the upstream activation of mtROS sensitive pathways, including but not limited to the MAPK pathway [34].Others reported AP1 activation in RAW 264.7 macrophages [50] and NF-κB activation in lymphoblastoid 244B cells [35] following subclinical radiation doses, indicating that the two transcription factors participate in the general cellular response to such insult.Upon activation, AP1 and NF-κB promote cancer cell migration though inducing the expression of many genes related to cell adhesion, cytoskeleton remodeling, matrix deposition and extracellular proteolysis [51,52].Interestingly, all mtROS [23], AP1 [53] and NF-kB [54] are positive EMT regulators in breast cancer cells, offering a likely molecular pathway to explain irradiation-induced EMT [17,18].
While the single delivery of a subclinical dose of radiation stimulated breast cancer cell migration, it did not trigger in vitro invasion, another necessary phenotype supporting metastasis.Yet, we previously showed that sustained mtROS production is a fundamental and essential characteristic of metastatic progenitor cells in human breast cancer models in mice [55].It is therefore possible that repeated subclinical dose delivery in fractionated radiotherapy regimen would eventually promote metastasis, which has been suggested by others based on clinical evidence [14].Exploring this possibility experimentally is a major perspective of our work.If verified, we believe that targeting mtROS could be a preferential therapeutic answer instead of targeting the numerous mitochondrial phenotypes capable of enhancing mtROS production and the multitude of redox-sensitive promigratory pathways downstream of mtROS.Among other drugs, MitoQ selectively inhibiting mtROS formation is a promising candidate as it already successfully passed phase I clinical trials with limited toxicity [56].For therapeutic mtROS inhibition, noninvasive mtROS imaging in tumors in vivo would also be useful.Specific probes developed for electron paramagnetic resonance bear this promise [57].
Conclusively, our study shows that breast cancer cell migration can be induced by a single subcytotoxic dose of photon irradiation, which can be prevented by mtROS inhibition.

Fig. 2
Fig. 2 Targeting ROS inhibits the human breast cancer cell migration induced by subclinical doses of irradiation.(A-C) MCF7 and MDA-MB-231 were irradiated or not with a single dose of 0.5 Gy and 0.125 Gy, respectively.(A) Where indicated, cells were treated with general antioxidant N-acetyl-L-cysteine (NAC, 4 mM) or mtROS inhibitor MitoQ (500 nM for MCF7 and 250 nM for MDA-MB231 cells) starting 24 h after irradiation and during a 24 h migration in transwells with FBS as chemoattractant.MCF7 cell migration is shown on the left (n = 4) and MDA-MB cell migration on the right graph (n = 7-8).(B) Twenty-four hours after irradiation, MCF7 and MDA-MB-231 cells were transfected with a plasmid encoding a mitochondria-targeted version of catalase (mtCAT).Mitochondrial H 2 O 2 measured 24 h later using MitoPY1 fluorescence is shown in the left graph for MCF7 (n = 4) and in the right graph for MDA-MB-231 (n = 5) cells.(C) Cells were transfected or not with a plasmid encoding mtCAT 1 h after irradiation, left to recover for 24 h, and then assayed for migration for 24 h in transwells with FBS as chemoattractant.Migration is shown in the left graph for MCF7 (n = 3-4) and in the right graph for MDA-MB-231 (n = 6) cells.All data are shown as mean ± SEM. * P < 0.05, ns P > 0.05, by Student's t test (A-C)

Fig. 3
Fig. 3 Enhancing H 2 O 2 generation in mitochondria triggers human breast cancer cell migration.(A) Cartoon (produced using BioRender) depicting the mtDAAO-HyPer system used to generate (DAAO reaction fueled by exogenous D-alanine) and detect (HyPer fluorescent reporter) H 2 O 2 selectively in cell mitochondria, based on previously reported data [29].The graph shows a standard curve (F 500 /F 420 HyPer fluorescence) generated 1 h after providing increasing doses of D-alanine to MCF7 cells expressing the mtDAAO-HyPer system, with 10 mM of L-alanine serving as a negative control (n = 5).(B-C) MCF7 and MDA-MB-231 were irradiated or not with a single dose of 0.5 Gy and 0.125 Gy, respectively, transfected with the mtDAAO-HyPer system 1 h later, and left to recover for 24 h before treatment with L-alanine (10 mM) or D-alanine (10 mM).(B) One hour later, HyPer fluorescence was measured in MCF7 (left graph, n = 5) and MDA-MB-231 (right graph, n = 5) cells.(C) After irradiation and transfection, MCF7 (left graph, n = 12) and MDA-MB-231 (right graph, n = 8-9) cell migration was determined over 24 h in transwells with FBS as chemoattractant in the presence of either L-alanine or D-alanine.All data are shown as means ± SEM. * P < 0.05, ** P < 0.01 compared to control; by one-way ANOVA with Dunnett post-hoc test (A) or by Student's t test (B, C)